Role of FAM20C in the Phosphorylation of SIBLING Protein

描述(由申请人提供)：这项研究计划重点关注FAM20C(也称为“牙本质基质蛋白4”或“DMP4”)在同胞蛋白磷酸化中的作用。我们最近的数据已经证实，FAM20C对于骨和牙齿的形成和矿化是必不可少的，这种蛋白激酶可能是负责催化兄弟姐妹蛋白磷酸化的主要酶。我们发现：1)FAM20C在骨、牙本质、牙骨质和釉质形成细胞中高表达；2)FAM20C失活导致软骨病小鼠骨骼广泛矿化不足；3)FAM20C-KO小鼠在牙本质、牙骨质和釉质的形成和矿化方面存在显著缺陷；4)FAM20C-KO小鼠骨或牙本质中非胶原蛋白的总磷酸化水平显著低于野生型小鼠。此外，我们和其他人发现，重组FAM20C在体外有效地磷酸化了一些兄弟蛋白。我们的初步数据使我们形成了一个中心假设，即FAM20C是催化同胞蛋白磷酸化的主要激酶，而Fam20C-KO小鼠硬组织中的缺陷可能是由于同胞蛋白磷酸化的显著丧失造成的。为了验证这一新假说，我们提出了以下三个具体目标：(1)确定FAM20C缺乏对同胞蛋白磷酸化的影响。我们将从Fam20C-KO小鼠的骨和牙本质中分离出OPN、BSP、DMP1的C端片段和DPP，并将这些蛋白的磷酸化水平与WT小鼠组织中的磷酸化水平进行比较。我们还将通过~(32)P掺入法检测FAM20C对来自Fam20C-KO和WT小鼠的颅骨成骨细胞培养中同胞蛋白磷酸化的影响。(2)用蛋白酶消化OPN、BSP、DMP1C-末端片段和DPP，分离蛋白水解肽，并对分离的多肽进行质谱测序，以检测Fam20C-KO和WT小鼠骨骼和牙本质中的同胞蛋白中的磷酸丝氨酸。(3)检测FAM20C-KO小鼠同胞蛋白对成骨细胞和成牙本质细胞矿化结节形成的影响。本申请中提出的多管齐下的方法将促进我们对FAM20C在同胞蛋白磷酸化中的作用的理解，并为进一步阐明与同胞蛋白磷酸化相关的生物矿化机制奠定基础。

DESCRIPTION (provided by applicant): This research proposal focuses on the role of FAM20C (also known as "dentin matrix protein 4" or "DMP4") in the phosphorylation of SIBLING proteins. Our recent data have established that FAM20C is essential to the formation and mineralization of bone and tooth, and that this protein kinase may be the primary enzyme responsible for catalyzing the phosphorylation of SIBLING proteins. We discovered: 1) FAM20C is highly expressed in the cells forming bone, dentin, cementum and enamel; 2) the inactivation of Fam20C in mice leads to rickets with extensive hypomineralization in murine bone; 3) Fam20C-KO mice have significant defects in the formation and mineralization of dentin, cementum and enamel; 4) the total phosphorylation level of non-collagenous proteins in the bone or dentin of Fam20C-KO mice is significantly lower than that of wild type mice. Furthermore, we and others revealed that recombinant FAM20C effectively phosphorylates some of the SIBLING proteins in vitro. Our preliminary data lead us to form the central hypothesis that FAM20C is the primary kinase catalyzing the phosphorylation of SIBLING proteins and the defects in the hard tissues of the Fam20C-KO mice may result from a significant loss of phosphorylation in the SIBLING proteins. To test this novel hypothesis, we propose the following three specific aims: (1) To determine the effects of FAM20C deficiency on the phosphorylation of SIBLING proteins. We will isolate OPN, BSP, the C-terminal fragment of DMP1, and DPP from the bone and dentin of Fam20C-KO mice and compare the phosphorylation level of these proteins with that in tissues from WT mice. We will also examine the effects of FAM20C on the phosphorylation of SIBLING proteins via 32P incorporation assay in the cultures of calvarial osteoblasts derived from Fam20C-KO and WT mice. (2) To examine phosphoserines in the SIBLING proteins isolated from the bone and dentin of Fam20C-KO and WT mice by digesting OPN, BSP, DMP1 C-terminal fragment, and DPP with proteases, separating the proteolytic peptides, and sequencing the isolated peptides with mass spectrometry. (3) To examine the effects of SIBLING proteins isolated from the Fam20C-KO mice on the mineralized nodule formation by osteoblasts and odontoblasts. The multipronged approaches proposed in this application will advance our understanding of the role of FAM20C in the phosphorylation of SIBLING proteins, and serve as the foundation for further elucidating the mechanism of biomineralization associated with the phosphorylation of SIBLINGs.